Proteolytic Labeling Methods

However, there are drawbacks to this approach, particularly the variability in the extent of 0 incorporation in the heavy -labeled peptides. This arises because of the possibility of back-exchange ( 0 replaces 0) when the peptide-trypsin complex is re-formed and subsequently hydrolyzed again. For this and other reasons this approach is not often used, since many precautions have to be taken in the acquisition and interpretation of the data (Stewart 2001). However, such labeling has been exploited (Shevchenko 1997) in experiments designed to assist in the interpretation of MS/MS spectra of peptides by distinguishing between primary fragments that contain the C-terminus (exhibit an appropriate mass shift) from N-terminal fragments (no such mass shift). 

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A recently developed method for lsO-based quantification of peptides uses catalysis with hydrochloric acid instead of standard proteases for labeling. Unlike proteolytic labeling, acid catalysis labels all carboxyl groups present in a peptide (Asp, Glu, carboxymethylated Cys, C-terminal carboxyl groups). Thus, most peptides labeled by this strategy have a mass difference >4 Da, making data analysis simpler and more reliable than conventional proteolytic labeling approaches (104). 

The proteomics research of a number of scientists was described in a C E News report of the 2001 Pittcon meeting.10 One group, that of Catherine Fenselau at the University of Maryland, has studied a new method for proteolytic stable isotope labeling to provide quantitative and concurrent comparisons between individual proteins from two entire proteome pools.11 Two lsO atoms are incorporated into the... 

Serum albumin labeled with an iodine radionuclide was firstly used as a substrate for determining protease activity by Absolon This method was later on modified several times and applied for assaying various proteolytic activities in different materials. Mego et al. injected denaturated I-human %rum albumin into the tail vein of rats and measured the rate of intralysosomal proteolysis on isolated lysosomes containing endocytosed substrate. This method was also used for the determining the intralysosomal pH on the basis of differences found in the rate of I-albumin breakdown in intact and lysed lysosomes C-bovine serum albumin, I-casein or I-albumin have been alternatively used as substrate for measuring the activity of trypsin, chymotrypsin and papain - ). 

Apart from assays of proteolytic activity and immunochemical methods (7.1 and 7.5), protein labeling is exploited in other areas of analysis. Noteworthy among them are protein determination in complex mixtures or even directly in cells, protein separation (affinity chromatography and electrophoresis) and protein detection following various separation techniques. For this purpose different labels are applied. 

A limited amount of information can be obtained by the use of proteolytic enzymes that detach either amino acids or dipeptides sequentially from the C-terminus. They are thus complementary to the aminopeptidases and dipeptidyl aminopeptidases. Two pancreatic enzymes, carboxypeptidases A and B, differ in specificity. The former preferentially liberates C-terminal amino acids with aromatic side chains, somewhat less readily amino acids with alkyl side chains and, more slowly still, other amino acids, but not Pro, Arg, Lys and His. In contrast, carboxypeptidase B releases only C-terminal Arg, Lys and His. Carboxypeptidase Y is much less specific and is capable of removing all amino acids, although Gly and Pro are liberated only slowly. As with aminopeptidases, it is advisable to analyse the hydrolysate at intervals in order to determine the C-terminal sequence of amino acids. An interesting recent development (Carles et al., 1988) uses carboxypeptidase to effect transpeptidation between the protein being sequenced and a tritiated amino acid. The labelled protein is then degraded by various specific methods and then the labelled fragments are isolated by gel electrophoresis and subjected to Edman degradation. 

Although sensitivity may be increased in many instances using polymer-based labels, others have reported that with some antibodies, particularly those that require proteolytic digestion or target unmasking, the EnVision method revealed slightly weaker staining and less sensitivity. This outcome was believed... 

One can place in some positions on the protein radioactive, fluorescent, or spin-labeled markers. For the same purpose one may also use antibodies and proteolytic enzymes. With these procedures one can determine those parts of the protein that are outside the membrane. Next, efforts are made to determine the position of the membrane part in the sequence. A complementary method is the hydrophobic marking of the protein by building into it hydrophobic photolabeled parts from the class of phospholipid compounds in order to determine those parts that are situated in the membrane. Another method uses proteolytic enzymes that split the protein in a specific position in the segment that is not located in the membrane. ... 

A fundamental limitation of the proteolytic fragmentation HX-MS method arises from the loss of the deuterium label (back-exchange) during immersion in the quench solution and throughout chromatographic analysis (see Section 2.3.8). When averaged across all analyzed peptides, the... 

Quantification by the Proteolytic 0-Water Labeling Approach This method, also known as enzymatic labeling, relies on trypsin-catalyzed labeling... 

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